Understanding the molecular pathways disrupted in motor neuron diseases is urgently needed. Here, we employed CRISPR knockout (KO) to investigate the functions of four ALS-causative RNA/DNA binding proteins (FUS, EWSR1, TAF15 and MATR3) within the RNAP II/U1 snRNP machinery. We found that each of these structurally related proteins has distinct roles with FUS KO resulting in loss of U1 snRNP and the SMN complex, EWSR1 KO causing dissociation of the tRNA ligase complex, and TAF15 KO resulting in loss of transcription factors P-TEFb and TFIIF. However, all four ALS-causative proteins are required for association of the ASC-1 transcriptional co-activator complex with the RNAP II/U1 snRNP machinery. Remarkably, mutations in the ASC-1 complex are known to cause a severe form of Spinal Muscular Atrophy (SMA), and we show that an SMA-causative mutation in an ASC-1 component or an ALS-causative mutation in FUS disrupts association between the ASC-1 complex and the RNAP II/U1 snRNP machinery. We conclude that ALS and SMA are more intimately tied to one another than previously thought, being linked via the ASC-1 complex.

In the genome, primary microRNAs (pri-miRNAs) are encoded either as independent transcriptional units with their own promoters (intergenic miRNAs) or within the introns of other genes (intronic miRNAs). Here, we report two methods, one that we established for coupled RNAP II transcription and pri-miRNA processing and the other that is a three-way system for RNAP II transcription, pri-miRNA processing, and pre-mRNA splicing. In these systems, CMV-DNA constructs encoding the processing substrates are incubated in HeLa cell nuclear extracts in the presence of P-UTP to generate the nascent RNAP II transcripts, which are processed efficiently by the endogenous RNA processing machineries in nuclear extracts.